With this protocol, a gas permeable chamber is created by means of a double-sided tape. The permeable chamber allows for a better preservation of the larval brains during imaging. The double-sided tape is porous (gas permeable) and hydrophobic (it prevents evaporation).

Time of mitosis will be an indicator of healthy brains

Figure 1. Under the dissection microscope, explant CNS from third instar larvae and isolate intact brains. Note: Fat bodies can be isolated as well to be added to the medium (see Cabernard 2013)

Figure 2. Stick a ring shaped double-sided tape on a square glass coverslip (high resolution, 24mm x 24mm) after removing both sides of paper from the tape. Note: optimal thickness of the double-sided tape for larval brains should be 1mm

Figure 3. Place a drop of 70µl of medium and gently place 5 to 10 brains. Place the brains with the desired orientation: the part to be observed must be facing the glass coverslip

Figure 4. Gently place the microfluidic thermalization chip on top of the chamber. It will stick to the tape and the medium will disperse across the chamber. Important: the chamber must be centered on the thermalization pattern of the microfluidic chip

Figure 5. Flip the mounted system 180°C. Brains will be facing up.

Figure 6. Remove the excess of medium by pinching a Pasteur pipette through the tape ring (porous). The brains must be in direct contact with the glass coverslip. Gently move the glass coverslip to maintain the brains in the desired orientation. Important: avoid excessive squeezing of brains (this can happen if too much medium is removed)

** For an inverted microscope configuration, carefully flip the mounted system 180°C once the brains are well oriented